World Journal of Pharmaceutical ReseaRch Mousa et al

www.wjpr.net Vol 3, Issue 3, 2014.

3704

Mousa et al. World Journal of Pharmaceutical Research

LEAF VOLATILES AND STEM BARK EXUDATES OF TWO

SWIETENIA SPECIES: COMPOSITION AND BIOACTIVITY Ola Mohamed Mousa*, Marwa Yousry Moustafa Issa, Hesham Ibrahim El-Askary

and Soheir Mohamed El Zalabani

Pharmacognosy Department, Faculty of Pharmacy, Cairo University,

Kasr-El-Aini Street, Cairo, Egypt, 11562

ABSTRACT

This study comprises a comparative physico-chemical investigation of

the hydrodistilled leaf volatiles and stem bark exudates of two

Swietenia species grown in Egypt viz., Swietenia mahogani (L.) Jacq.

and Swietenia macrophylla King. The physical characters were

described, and chemical composition determined via chromatographic

analyses (PC, GLC and GC/MS). Moreover, the antimicrobial potential

of all samples was assessed and the long-term antihyperglycemic

activity of gum exudates evaluated. Hydrodistilled leaf volatiles (0.15

vs. 0.10% v/dry wt. in S. mahogani and S. macrophylla, respectively)

were dominated by sesquiterpenoids among which hydrocarbons

prevailed (75.51 vs. 80.95%), as evidenced by GC/MS analysis. Trans-

caryophyllene (33.89%) dominated the S. mahogani sample and α-

humulene (39.64%) that of S. macrophylla. Oxygenated constituents were minor in both,

being mainly represented by sesquiterpenoids, with elemol (6.13%) as major in S. mahogani

and E-nerolidol (10.18%) in S. macrophylla. Analytical parameters (moisture content and

total ash) of the exudates and mineral composition of ashes were determined. GLC analysis

of the sylilated exudate hydrolysates revealed that galactose dominated the sugar composition

of the samples (57.99 vs. 59.57%) followed by xylose (8.24 vs. 8.37%). In addition, traces of

glucuronic acid were detected in both samples. The volatiles were found effective against all

tested Gram-positive bacteria meanwhile stem bark exudates inhibited mycobacterial growth

only, and yeast was not affected by any of the samples. Minimum inhibitory concentrations

were determined. A significant reduction in blood glucose level was recorded in Alloxan-

diabetic rats treated with the aqueous solutions of the stem bark exudates of both species.

World Journal of Pharmaceutical ReseaRch

Volume 3, Issue 3, 3704-3722. Research Article ISSN 2277 – 7105

Article Received on 25March 2014, Revised on 08 April 2014, Accepted on 21 April 2014

*Correspondence for

Author

Dr. Ola Mohamed Mousa

Pharmacognosy

Department, Faculty of

Pharmacy, Cairo

University, Kasr-El-Aini,

Street, Cairo, Egypt,

11562


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Key words: Swietenia species, leaf volatiles, stem bark exudates, antimicrobial,

antihyperglycemic.

INTRODUCTION

Swietenia species (Meliaceae) constitute a small genus of tropical American forest trees.

Swietenia mahogani (L.) Jacq. provides the original "American mahogany" wood; supplies

have, however, become very rare due to over-harvesting and the majority of the trade is

currently from the faster growing Swietenia macrophylla King [1, 2]. Both species are, as well,

grown for shade and ornament [1-4] and were, in this respect, naturalized in Egypt. The genetic

and botanical profiling of the two Egyptian plants indicated a relatively high degree of

taxonomical similarity, despite providing distinct criteria for discrimination [5]. Limonoids

isolated from various meliaceous are reputed as potent insect antifeedants and growth

regulators [6]. The low toxicity of these natural products to non-targeted organisms has

prompted extensive trials for their isolation [6]. Secondary metabolites of certain American

and Asian Swietenia spp. have been extensively investigated as a source of useful non-wood

forest products, especially the antifeedant tetranortriterpenoids [6] and the seed oil [7].

Swietenia seeds are traditionally used as antihypertensive, antidiabetic and antimalarial; the

stem bark decoctions are, in addition, taken as potent febrifuge and antidiarrheal, and applied

as wound astringent [1-2, 4]. Furthermore, the acaricidal activity of the ethanol extracts of the

leaves and stem bark of the locally cultivated Swietenia mahogani and Swietenia macrophylla

were tested against Varroa destructor mite, a parasite with marked economic impact on the

beekeeping industry [8]. A pronounced miticidal activity was noticed without almost

affecting the bees; thus suggesting the use of either the plants extracts or products derived

there from as valuable ecofriendly biodegradable agents for controlling Varroa mite [8].

The scarcity of reports concerned with the leaf volatiles and stem bark exudates of the

selected species stimulated the performance of this study. The present comparative

investigation aimed to throw light on the composition of the leaf volatiles and stem bark

exudates of the locally cultivated Swietenia mahogani and Swietenia macrophylla in view to

provide possible chemotaxonomical criteria for interspecies differentiation. In addition, the

antimicrobial potential of these plant products and anti-hyperglycemic activity of the

exudates was evaluated intending further implementation in national drug industry.


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MATERIALS AND METHODS

Plant material

Leaves and stem barks of Swietenia mahogani (L.) Jacq., and Swietenia macrophylla King.

were obtained from plants cultivated at the Zoological Garden, Giza, Egypt. Identification of

the samples was kindly confirmed by Dr. Mohamed El-Gebaly, botanist specialist and

voucher specimens kept at the Herbarium of the Pharmacognosy Department, Faculty of

Pharmacy, Cairo, Egypt.

Leaf volatiles

Fresh leaves (1kg) were gathered in January (winter) from of each of the plants under

investigation. Each sample was individually subjected to hydrodistillation in a Clavenger's

apparatus [9]. The isolated volatiles were dried over anhydrous sodium sulfate and samples

saved in a refrigerator for further analysis.

Stem bark exudates

The dried exudates were collected after a lapse of three weeks from incisions made in the

barks of the plants, at the trunk level and saved for chemical and biological investigation.

Microorganisms and experimental animals

A set of bacterial and fungal strains (available in stock cultures at the Microbiology

Department, Faculty of Pharmacy, Cairo University) was used for evaluation of the

antimicrobial activity. This comprises Staphylococcus aureus (ATCC 4175), Sarcina lutea

(Laboratory collection strains) and Bacillus subtilis (NCTC 6633) as representative Gram-

positive bacteria; Escherichia coli (ATCC 10536), Proteus vulgaris (NCTC 4175) and

Pseudomonas aeruginosa (CNCM A21) as Gram-negative ones; and Mycobacterium phlei

(Laboratory collection strains) as a type of acid fast bacilli. The yeast Candida albicans

(ATCC 60193) was the tested fungal strain. Adult male albino rats of Sprague Dawley strain

(120-150 g, obtained from the animal house colony at the National Research Centre, Guiza,

Egypt) were utilized for assessment of the long-term anti-hyperglycemic activity. The

animals were kept on standard laboratory diet composed of: vitamin mixture (1%), mineral

mixture (4%), corn oil (10%), sucrose (20%), cellulose (0.2%), casein (10.5%) and starch

(54.3%). Water was supplied ad. Libitum. All the animal procedures were carried out

according to the agreement of the Ethics Committee of The National Research Centre, Egypt

and in harmony with the recommendations of the proper Care and Use of Laboratory

Animals.


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Reference samples, solvent systems and chemicals

Authentic reference sugars used for PC and GLC analysis of the hydrolysates of the stem

bark exudates were purchased from E-Merck, (Darmstadt, Germany). Solvent systems used

for PC were: n-butanol-pyridine-water 6:4:3 v/v (S1) and n-butanol-acetic acid-water 4:1:5

v/v (S2). All chemicals utilized in this study were of analytical grade.

Drugs and kits

Ofloxacin and Amphotericin B (Bristol-Myers Squibb, Switzerland) were utilized as standard

antibacterial and antifungal, respectively. Alloxan (Sigma, USA) solution (10 mg/0.1 ml) was

used by intraperitoneal route for induction of diabetes; Metformin (Cidophage®, Chemical

Industries Development Co., CID Co., Giza, Egypt) was used as standard antidiabetic and

Bio-Merieux kits were employed for measuring blood glucose levels (Bio-Merieux Co,

France).

Characterization and GC/MS analysis of the hydrodistilled volatiles

The percentage yield of the hydrodistilled volatiles was calculated on dry weight basis, and

organoleptic characters described. The samples were then subjected to chromatographic

analysis on a GC/MS system (Hewlett Packard G1800A GCD coupled to an HP automatic

injector 7673A) operated in an electron impact mode. Separation was achieved on an HP-5-

MS capillary column (30 m 0.25 mm, 0.25m film thickness) by adopting the following

conditions: injector temperature, 220oC; electron ionization detector temperature, 280oC;

carrier gas, He (1 ml/min); oven temperature program: initial temperature 40oC, increased to

160oC at a rate of 4oC/min, isotherm for 3 min, increased to 280oC at a rate of 10oC/min and

kept isotherm for 4 min i.e. ramp function programming. Mass spectra were taken at 70eV.

Mass range was from m/z 40-500. Library search was carried out using a Willey 275 L GC-

MS data base. A series of authentic n-alkanes (C8-C22, Poly Science Inc., Niles, USA) was

subjected to GLC analysis under the same experimental conditions and the retention indices

(Kovat's indices, KI) of the oil constituents computed by logarithmic interpolation between

bracketing alkanes. Identification of individual components was confirmed by comparison of

their retention indices and MS fragments patterns with published data [10, 11]. Relative

percentage amounts were calculated from the Total Ion Chromatograms by a computerized

integrator.


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Characterization of the stem bark exudates

Organoleptic characteristics (condition, color and odor) of the exudates were examined. The

solubility in different solvents as well as distilled water was determined at 25°C, and optical

activity (of 1% solution in distilled water) was measured, at 25°C in a 1 dm tube using a

Polyscience Div. Preaton Ind. Inc polarimeter. The two samples were then subjected to

chemical tests for carbohydrates, proteins, tannins and oxidase enzymes [9, 12-14]. Analytical

parameters including moisture and total ash contents were determined in triplicates [15, 16].The

moisture content (expressed as %) was determined after heating 1gm samples of air-dried

exudates in an air-oven at 120 °C for 2 hours followed by keeping in a desiccator till constant

weight. The total ash content (calculated as %) was determined by gradual heating the oven-

dried samples (1 gm, each), in an ignition crucible, up to 1100°C then temperature

maintained isothermal at 1100°C for at least 1 hour.

Determination of the mineral composition of the stem bark exudates

The mineral composition of the acid-soluble and acid-insoluble ashes of each of the two

exudates was determined. The cationic components of the acid-soluble ash were estimated in

the filtrates obtained upon boiling weighed amounts of the total ash samples for 3 to 5 min in

1:1 HNO3; the analysis was performed by atomic absorption spectrometry (at 800 °C) in a

Perkin Elmer 2380 atomic absorption spectrometer, equipped with an acetylene-air flame.

The mineral content of the acid-insoluble ash was determined gravimetrically (as µg silicon/g

ash) after incineration of the nitric-acid insoluble residue in an ignition crucible at 1100°C, as

processed for total ash determination.

Analysis of the sugar composition of the stem bark exudates

The monosaccharide composition of the acid-hydrolysates of the exudates was analysed by

both paper and gas-liquid chromatography (PC and GLC).

Preparation of the samples: For PC; aliquots (0.5 gm) of the exudates were hydrolysed by

heating with 2N H2SO4 (boiling water bath for 24 hours), hydrolysates filtered purified by

treatment with BaCO3 and monosacharides extracted from the dried filtrates with hot

pyridine, freed from the solvent then redissolved in 10% isopropanol to be used as spotting

liquids. For GLC; samples (0.1 gm) were heated with 1N HCl (10 ml), for 5 hours on a

boiling water bath [17]; the neutralized hydrolysates (0.5 ml, each) were evaporated to dryness

under a stream of nitrogen at 40oC, in a small screw-stopped septum vial, 0.5 ml isopropanol

was then added to each sample and the solvent completely removed under a stream of


3709


nitrogen; the septum was then screwed on and 0.5 ml of 2.5% hydroxylamine hydrochloride

in pyridine injected into the vial; the resulting solution was mixed, heated for 30 minutes at

80oC, then allowed to cool; silylation of the hydrolysates was performed by using a mixture

of trimethylchlorosilane and N,O-bis-(trimethylsilyl) acetamide, 1:5 v/v, the silylating

reagent (1 ml) was injected in the sample solution, mixed, heated for 30 minutes at 80oC and

then cooled; for GLC analysis, samples (1l, each) of the silylated hydrolysates were

used [18].

Chromatographic analysis: PC of the hydrolysates was performed on Whatmann No. 1

sheets alongside with available authentic sugars (development technique, ascending; solvent

systems, S1 and S2; visualization, aniline phthalate spray reagent [19] and heated at 105oC for 5

min). GLC of silylated hydrolysates was performed on a Hewlett-Packard HP 6890 N

network GC system equipped with a ZB-1701 capillary column (30 m 0.25 mm , 0.25 m

film thickness) and conducted under the following operating conditions: injector

temperature, 250° C; FID detector, temperature 270oC, air flow rate 45 ml/min, H2 flow rate

40 ml/min; carrier gas, He (1.2 ml/min); oven temperature program: initial temperature

150oC, isotherm for 2 min, increased to 200oC at a rate of 7oC/min, then kept isotherm for 20

min. Identification of the components was based on comparison of their retention times with

those of authentic samples similarly analyzed.

Assessment of the antimicrobial activity

The antimicrobial activity of the hydrodistilled volatiles and stem bark exudates, was tested

against the selected bacterial and fungal strains. The Minimum Inhibitory Concentrations

(MICs) of the samples exhibiting significant activity against specific strains were further

determined. The agar diffusion method from cups [20, 21] was adopted for evaluation of the

antimicrobial activity. Tripticase soy agar (Difco) was used as culture medium. Cups (0.5 cm

in diameter) were made using a no.3 cork borer. The samples were dissolved in DMSO at a

concentration of 100 mg/ml for each the volatiles and stem bark exudates. Aliquots of 50 l

of each of the tested samples (equivalent to 5 mg) were, separately, aseptically added to the

cups of the inoculated plates (previously prepared). The plates were incubated while inverted,

at 37°C for 24 hours in case of bacteria and at 25°C for 48 hours in case of fungi (yeasts).

DMSO (50 l) was used as a negative control and cups of Ofloxacin and Amphotericin B (5

µg/cup, each), were used as positive controls. After incubation, zones of inhibition were

measured and diameters less than 5 mm were considered as an indication of no growth


3710


inhibitory effect. The percentage potency as compared to the appropriate reference drug was,

in each case, calculated. Minimum inhibitory concentrations (MIC) were determined using

the dilution method [20]. Several dilutions of each active sample were incubated, as previously

described, with each of the microorganisms towards which it exhibited a significant growth

inhibitory effect. A curve representing the relationship between the bacterial count

(colony/ml sample) and the concentration of the sample (l/ml) was plotted and minimum

inhibitory concentrations deduced.

Assessment of the long-term anti-hyperglycemic activity

Diabetes was induced to male albino rats of Sprague Dawley strain (120-150 g) by

intraperitoneal injection of Alloxan (150 mg/kg b.wt.), as described by Eliasson and Samet [22]. The experimental animals were divided in four groups, each of 10 animals. Samples of

the stem bark exudates and standard antihyperglycemic drug, Metformin (150 mg/kg b.wt.,

each) were administered orally, followed by collection of blood samples, at intervals, for

determination of blood glucose levels. The long-term anti-hyperglycemic activity was

evaluated adopting the method described by Trainder [23]. Glucose levels were measured in

blood samples collected at zero time (G0, prior treatment) and after 4 and 8 weeks intervals

from administration of the tested samples (in appropriate doses) in case of treated animals

(Gt). The percentage change in blood glucose level from initial glycemia was, in each case,

calculated according to the following equation: % of change = (G0–Gt) × 100/G0.

The data obtained were analyzed using student's t- test where means of the treated groups

were compared to that of the control group for each variable [24].

RESULTS AND DISCUSSION

Yield, physical characteristics and composition of the leaf volatiles

The volatiles isolated by hydrodistillation from fresh leaves of Swietenia mahogani (L.) Jacq.

amounted to 0.02% v/w (calculated on dry weight basis); being higher in those of Swietenia

macrophylla King., reaching 0.03 % v/w. The two samples exhibited nearly the same

physical characters being oily, pale yellow in color, with a characteristic woodsy balsamic

aromatic odor and readily soluble in ethanol 70%. Components identified by GC/MS analysis

of the isolated volatiles, their Kovat's indices, relative percentages and mass spectral data are

listed in tables (1 and 2) and represented in figs. (1-3).

Data of GC/MS analysis revealed a qualitative and quantitative variability in composition

between the examined volatiles. The total number of constituents identified under the adopted


3711


operating conditions was 42 among which 18 components were common in the two samples.

Components identified were 27 in number in the volatiles of S. mahogani and 33 in S.

macrophylla representing 95.54% vs 96.88% of the total composition. Hydrocarbons

dominated the chromatographic profiles of the two volatiles (76.63% vs 82.75% in S.

mahogani and S. macrophylla, respectively) with prevalent sesquiterpenoids (75.51% vs.

80.95%). Trans-caryophyllene was the major in S. mahogani (33.89%), reaching only

(29.12%) in S. macrophylla being exceeded by α-humelene (39.64%) in that sample.

Oxygenated constituents were minor in both S. mahogani and S. macrophylla (18.91% vs

14.13%, respectively) and are mainly sesquiterpenoid in nature (18.24% vs 13.03%).

Alcohols (12.35% vs 10.45%) were prevalent with major elemol in S. mahogani (6.13 %) and

E-nerolidol in S. macrophylla (10.18%). Oxides were detected in appreciable amounts

(5.89% vs 2.68%). The variability in composition among the volatiles of these two closely

related species could serve as a helpful tool for chemotaxonomical discrimination. The oils

appeared to be rich in insect attracting pheromones such as trans-caryophyllene and α-

humulene [25]. As a matter of fact, the prevalence of sequiterpenoid hydrocarbons in the

volatiles of the leaves of S. macrophylla was previously reported (major component

germacrene D, 58.5-66.5%) [26]; yet, a noticeable qualitative variation is, here, recorded for

the Egyptian sample. This may be attributed to climatic and/or geographical factors.

Table (1): Identified components in the hydrodistilled volatiles of the leaves of Swietenia mahogani and Swietenia macrophylla

Peak # Identified Component KI Adams

Relative percentage (Observed KI)

M+ B S. mah. S. macr.

1 n-Decane 999 0.52 (990) 0.57 (989) 142 57

2 n-Octanal 1001 0.21 (996) — 128 41

3 Limonene 1031 0.17 (1020) — 136 67

4 n-Undecane 1099 0.05 (1085) 0.12 (1084) 156 57

5 n-Nonanal 1102 0.33 (10930 0.21 (1094) 142 57

6 Pinocarvone 1162 — 0.36 (1155) 150 53

7 trans-β-Terpineol 1163 — 0.10 (1163) 154 43

8 (Z)-3-Hexenyl butyrate 1186 0.13 (1179) 0.28 (1178) 170 67

9 n-Dodecane 1199 — 0.22 (1190) 170 57

10 Myrtenyl acetate 1235 — 0.15 (1230) 194 43

11 n-Tridecane 1299 — 0.14 (1287) 184 57


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12 δ-Elemene 1339 0.27 (1337) 0.03 (1331) 204 93

13 α-Cubebene 1351 — 0.13 (1335) 204 105

14 α-Copaene 1376 — 3.82 (1371) 204 105

15 β-Bourbonene 1384 2.06 (1378) 1.63 (1379) 204 81

16 cis-Caryophyllene 1404 1.20 (1400) 0.83 (1398) 204 41

17 trans-Caryophyllene 1418 33.89 (1416) 29.12 (1415) 204 41

18 α-Humulene 1454 6.56 (1459) 39.64 (1460) 204 93

19 γ-Muurolene 1477 — 0.14 (1473) 204 161

20 Germacrene D 1480 26.77 (1486) 0.71 (1484) 204 161

21 β-Selinene 1485 — 1.18 (1485) 204 93

22 cis-β-Guaiene 1490 2.15 (1495) — 204 105

23 α-Farnesene (E,E) 1508 — 2.87 (1504) 204 93

24 γ-Cadinene 1513 — 0.73 (1515) 204 161

25 δ-Cadinene 1524 2.38 (1526) 0.12 (1529) 204 119

26 α-Cadinene 1538 0.23 (1534) — 204 105

27 Elemol 1549 6.13 (1555) — 222 59

28 E-Nerolidol 1564 — 10.18 (1566) 222 41

29 β-Caryophyllene oxide 1581 5.31 (1585) 1.58 (1581) 220 41

30 Humulene epoxide II 1606 0.58 (1610) 1.10 (1607) 220 67

31 10-epi-γ-Eudesmol 1619 0.91 (1630) — 222 161

32 t-Muurolol 1641 1.18(1650) 0.13 (1646) 222 43

33 Torreyol 1645 — 0.04 (1650) 222 161

34 α-Cadinol 1653 3.72 (1661) — 222 43

35 Khusinol 1674 0.41 (1683) — 220 41

36 Heptadecane 1700 — 0.23 (1698) 240 57

37 n-Octadecane 1800 0.11 (1796) 0.17 (1797) 254 57

38 n-Nonadecane 1900 0.07 (1896) 0.16 (1892) 268 57

39 n-Eicosane 2000 — 0.07 (1995) 282 57

40 n-Heneicosane 2100 0.09 (2097) 0.07 (2096) 296 57

41 n-Docosane 2200 0.07 (2195) — 310 57

42 n-Tricosane 2300 0.04 (2298) 0.05 (2295) 324 57 Total number of identified constituents 27 33 Total percentage of identified constituents 95.54 96.88

S. mah.: Swietenia mahogani (L.) Jacq. (Volatiles of the leaves) S. macr.: Swietenia macrophylla King. (Volatiles of the leaves) KI Adams: Kovat's indices according to Adams (1995); Observed KI: Observed Kovat's indices M+: molecular weight. B: base peak


3713


Table (2): Relative percentages of the different classes of constituents identified in the volatiles of the leaves of Swietenia mahogani and Swietenia macrophylla

Constituents Relative percentage S. mah. S. macr.

Non-oxygenated constituents (Hydrocarbons):

Aliphatics 0.95 1.8

Monoterpenoids 0.17 0

Sesquiterpenoids 75.51 80.95 Total non-oxygenated constituents 76.63 82.75 Oxygenated constituents:

Aliphatics 0.67 0.49 Monoterpenoids 0 0.61 Sesquiterpenoids 18.24 13.03 Total oxygenated constituents 18.91 14.13 Alcohols 12.35 10.45 Aldehydes 0.54 0.21 Ketones 0 0.36 Esters 0.13 0.43 Oxides 5.89 2.68

S. mah.: Swietenia mahogani (L.) Jacq. (Volatiles of the leaves)

S. macr.: Swietenia macrophylla King. (Volatiles of the leaves)

0.52

0.05

0.11

0.070.09

0.04

0.57

0.12

0.17 0.16

0.070.05

0

0.1

0.2

0.3

0.4

0.5

0.6

n-Decane n-Undecane n-Octadecane n-Nonadecane

n-Heneicosane

n-Tricosane

Perc

enta

ge

S. mahoganiS. macrophylla

Fig. (1): Histogram representing the quantitative variability among the aliphatic

hydrocarbons identified in the volatiles of the leaves of Swietenia mahogani and

Swietenia macrophylla


3714


0.272.06 1.2

33.89

6.56

26.77

00.031.63 0.83

29.12

39.64

0.71 0.730

10

20

30

40

cis-C

aryop

hylle

netra

ns-C

aryop

hylle

ne

Germac

rene D

Perc

enta

ge

S. mahoganiS. macrophylla

Fig. (2): Histogram representing the quantitative variability among the sesquiterpenoid

hydrocarbons identified in the volatiles of the leaves of Swietenia mahogani and


0.21 0.13

5.31

0.58

1.18

00.28

1.58

1.1

0.13

0

1

2

3

4

5

6

n-Octanal (Z)-3- Hexenylbutyrate

β-Caryophylleneoxide

Humuleneepoxide II

t-Muurolol

Perc

enta

ge

S. mahogani

S. macrophylla

Fig. (3): Histogram representing the quantitative variability among the oxygenated

constituents identified in the volatiles of the leaves of Swietenia mahogani

andSwieteniamacrophylla

Characterization and analysis of stem bark exudates

The exudates collected after incision of the stem bark of the two locally cultivated species

were obtained as nearly odorless solids usually polyhedral in shape; being transparent and

light yellow in case of S. mahogani, and more or less translucent and amber-colored in case

of S. macrophylla. The analytical parameters (solubility in H2O, optical activity, moisture and

ash contents) and response to chemical tests are listed in table (3).


3715


Table (3): Analytical parameters and response to chemical tests of the exudates of

Swietenia mahogani and Swietenia macrophylla

Analytical parameters / Tested constituents

Stem bark exudates S. mahogani S. macrophylla

Solubility in H2O ( 25oC) 1: 83 1: 104 Optical activity (25oC) + 8.5 + 11.6 Moisture content (%) 5.02 6.64 Ash content (%) 1.39 0.53 Volatiles Not detected Not detected Carbohydrates Detected Detected Oxidase enzymes Detected in traces Detected in traces Proteins Detected in traces Detected in traces Tannins Detected in traces Detected in traces

The exudates were found soluble in water (dextrorotatory solution), but insoluble in alcohol,

ether or chloroform. They gave similar response to chemical tests indicating their

carbohydrate nature. Proteins, tannins and oxidase enzymes were detected in traces, while

hydrodistillable volatiles were absent. The analytical parameters were however different. The

mineral composition of the acid-insoluble ash as determined by Atomic Absorption

Spectrometry (cations concentrations, µg/g ash) is displayed in table (4).

Table (4): Mineral composition of the acid-soluble ash of the exudates of Swietenia

mahogani and Swietenia macrophylla

Cations Concentration (µg/g ash , 800°C)

Swietenia mahogani Swietenia macrophylla Calcium 683130 225510 Chromium Nil Nil Iron 21430 9680 Magnesium 60260 237830 Manganese 720 610 Potassium 63200 376860 Sodium 93330 372270 Zinc 70 458

Results of table (4) revealed the absence of Cr in both samples. Meanwhile, Zn was detected

in the lowest amount and Ca in the highest. In addition, Na concentration exceeded that of K

in the S. mahogani exudate, the order being reversed in S. macrophylla. The mineral content


3716


of the acid-insoluble ash (gravimetrically determined in terms of µg/g Silicon) was higher

(620) in S. macrophylla than in S. mahogani (450).

Results of PC analysis of the sugar components of the exudate hydrolysates as compared to

authentic samples are represented in table (5). Meanwhile, those obtained on GLC analysis of

the silylated derivatives of the hydrolysates are represented in table (6). The sensitivity of the

GLC technique is obvious since it allowed the identification of a larger number of

components as compared to PC (7 vs. only 3), Galactose, xylose and rhamnose being

detected by both techniques. The components identified under the GC analytical conditions

adopted reached about 73% of the total composition in the two samples; where galactose was

detected as the major followed by xylose, while glucuronic acid was the minor. Relative

amounts of all components were almost similar in the two hydrolysates except for arabinose

which was higher in the S. macrophylla sample and L-rhamnose in that of S. mahogani.

Table (5): Results of PC analysis of the exudate hydrolysates of Swietenia mahogani and


Authentic samples

Rf values Color with aniline phthalate

Exudate hydrolysates

S1 S2 S. mahogani S.macrophylla

L-Rhamnose 0.63 0.37 Yellowish-brown –

Xylose 0.52 0.28 Reddish-violet + + Arabinose 0.48 0.25 Reddish-brown – – Fructose 0.46 0.20 Brown – – Mannose 0.43 0.19 Brown – – Glucose 0.40 0.23 Brown – – Galactose 0.36 0.17 Pale brown + + Glucuronic acid 0.1 0.15 Pale brown – – Galacturonic acid 0.06 0.12 Pale brown – –

S1 n-butanol-pyridine-water 6:4:3 v/v , S2 n-butanol-acetic acid-water 4:1:5 v/v, (+):

detected, (): faint, (-): not detected


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Table (6): Components identified by GLC in the exudate hydrolysates of Swietenia

mahogani and Swietenia macrophylla

Identified compounds RRt (min) Relative % in exudate hydrolysates

S. mahogani S. macrophylla Xylose 0.65 8.24 8.37 Arabinose 0.67 1.05 1.81 Ribose 0.69 0.02 0.03 L-rhamnose 0.75 3.66 0.92 Galactose 1 57.99 59.57 Glucose 1.01 1.57 1.46 Glucuronic acid 1.32 0.56 0.42

RRt= Retention time relative to galactose (Rt=11.29 min)

The monosaccharide composition of the hydrolysates seemed in agreement with previous

data on the constitution of the polysaccharide of the gum exudates of Swietenia species

growing abroad which was reported to be mainly formed of galactose [27, 28].

Anti-microbial activity

The growth inhibitory activity of the leaf volatiles on the selected bacterial and fungal strains

(diameter of zone of inhibition and % potency) are displayed in table (7) and MIC values

listed in table (8).

Table (7): Antimicrobial activity of the volatiles of the leaves of Swietenia mahogani and


Tested Microorganisms Diameter of zone of inhibition (mm)

(% Potency)* S. mah. S. macr. Ofx. Amp. B

Escherichia coli ATCC 10536

-

- 29 (100) - Proteus vulgaris NCTC 4175 - - 38(100) -

Pseudomonas aeruginosa CNCM A21 - - 29(100) - Staphylococcus aureus ATCC 4175 25(78.13) 20(62.5) 32(100) - Sarcina lutea** 20(66.67) 17(56.67) 30(100) - Bacillus subtilis NCTC 6633 11(30.56) 12(33.33) 36(100) - Mycobacterium phlei** 20(80) 17(68) 25(100) - Candida albicans ATCC 60193 -

- - 25(100)

*Percentage of Potency as compared to standard drug; **: Laboratory collection strains S. mah.: Swietenia mahogani (L.) Jacq. sample S. macr.: Swietenia macrophylla King. sample Ofx.: Ofloxacin (5µg/cup) Amp. B: Amphotericin B (5µg/cup); - : no inhibition zone


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Table (8): Minimum inhibitory concentrations (MIC, l/ml) of the volatiles of the leaves

of Swietenia mahogani and Swietenia macrophylla

Tested Microorganisms MIC (l/ml)

S. mah. S. macr. Staphylococcus aureus ATCC 4175 6 6 Sarcina lutea 6 12.5 Bacillus subtilis NCTC 6633 12.5 12.5 Mycobacterium phlei 6 12.5

S. mah.: Swietenia mahogani (L.) Jacq. sample

S. macr.: Swietenia macrophylla King. Sample

A significant antibacterial activity was observed for the two volatiles against the tested Gram-

positive bacteria and the acid-fast Mycobacterium phlei, while no effect was recorded on

either the selected Gram-negative rods or the fungus Candida albicans; MICs of the volatiles

against Gram-positive bacteria as well as Mycobacterium phlei (table 8) varied from 6-12.5

µl/ml, indicating a high antibacterial activity against these microorganisms. Meanwhile, the

two stem bark exudates revealed only a moderate anti-mycobacterial activity while failing to

exert any effect on the other tested microorganisms.

Long-term anti-hyperglycemic activity

Results obtained on assessing the long-term anti-hyperglycemic effect (table 9) revealed a

significant reduction in blood glucose level in Alloxan-diabetic rats treated with aqueous

solutions of the stem bark exudates. The aqueous extract of the stem bark exudate of

Swietenia mahogani, orally given at a dose of 150 mg/kg b.wt., exhibited a slightly higher

activity than that of Swietenia macrophylla as compared to the standard drug Metformin

administrated at the same dose level (69 vs 62% potency).


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Table (9): Long term antihyperglycemic activity of the aqueous solutions of the stem bark exudates of Swietenia mahogani and Swietenia macrophylla in diabetic rats (n=10).

Animal group (n=10)

Blood glucose level (mg/dl)

Potency Zero time After 4 weeks After 8 weeks

Mean±S.E. Mean±S.E. % change Mean±S.E. %

change Diabetic 261.4±9.8 265.6±11.3 — 271.5±13.2 — —

S. mah. exudate 251.3±11.2 173.4±5.1* 31 134.2±5.8* 46.6 0.69

S. macr. exudate 262.8±14.5 192.6±7.3* 26.7 153.7±6.1* 41.5 0.62

Metformin 150 mg/kg b.wt. 265.9±12.8 147.1±6.2* 44.7 87.4±3.5* 67.1 1

Statistically significant from the control at p<0.1 S.E. = standard error

% of change is calculated as regards to the control group

S. mah.: Swietenia mahogani aqueous solution; S. macr.: Swietenia macrophylla aqueous

solution

CONCLUSION

Studies on the Egyptian Swietenia cultivars were mainly of agrochemical interest and focused

on isolation and establishment of structure-activity relationship of their insecticidal limonoid

components. The objective of this work was targeted towards assessing the efficacy of the

locally cultivated Swietenia mahogani (L.) Jacq. and Swietenia macrophylla King., as a

source of potential medicinals in order to further increase their propagation. The yield of

hydrodistilled leaf volatiles was found higher in S. mahogani than S. macrophylla. Non-

oxygenated and oxygenated sesquiterpenoids were prevalent with hydrocarbons constituting

the major make up of the oils. Moreover, the oils appeared to be rich in insect attracting

pheromones such as Trans-caryophyllene (in S. mahogani) and α-humulene (in S.

macrophylla). The apparent variability in composition could provide useful taxonomical

criteria for interspecies differentiation. The physico-chemical characteristics of the stem bark

exudates were established including organoleptic features, analytical parameters, and mineral

and carbohydrate composition. The exudates were devoid of Cr, but rich in Ca and galactose

and contained traces of glucuronic acid. The volatiles were found effective against a set of

Gram-positive bacteria while stem bark exudates inhibited mycobacterial growth only and

yeast was not affected by any of the samples. A significant reduction in blood glucose level


3720


was recorded in Alloxan-diabetic rats treated with the aqueous solutions of the stem bark

exudates of both species.

ACKNOWLEDGEMENT

The authors are indebted to Dr. Amani A. Sleem, Professor of Pharmacology, National

Research Center (NRC, Guiza, Egypt) for her kind help during the evaluation of the long-

term antihyperglycemic activity of the plant products.

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